We here describe the stereochemical aspects of the reactions of pyridoxal 5'-phosphate (PLP)-dependent enzymes, and the relationship between the stereochemistry of the enzyme reaction and molecular evolution of the enzyme. The reactions of PLP-dependent enzymes proceed through the formation of an anionic Schiff base intermediate between the substrate and the coenzyme. Three stereochemical possibilities exist for the formation and cleavage of bonds in the intermediate: the reaction occurs stereospecifically on either the si- or the re-face of the planar intermediate, or alternatively, non-stereospecifically on both faces. The stereospecificities for hydrogen transfer between C-4' of the cofactor and substrate in the transamination catalyzed by various PLP-dependent enzymes have been studied. The stereospecificities reflect the active-site structures of the enzymes, especially the topographical situation of a coenzyme-substrate Schiff base and a catalytic base for the hydrogen transfer. The aminotransferases and other PLP-enzymes catalyzing the transamination as a side-reaction so far studied catalyze only the si-face specific hydrogen transfer. This suggests that these PLP enzymes have similar active-site structures and are evolved divergently from a common ancestral protein. We recently established a new method for the identification of stereospecificity for the hydrogen transfer, and found that D-amino acid aminotransferase and branched chain L-amino acid aminotransferase, which have significant sequence similarity to each other, catalyze the re-face hydrogen transfer on the intermediate. The X-ray crystallographic studies of D-amino acid aminotransferase showed that the relative arrangement of the catalytic base of the enzyme active center to the C4' of the bound cofactor is opposite to that of other aminotransferases catalyzing the si-face hydrogen transfer. The folding of D-amino acid aminotransferase is also different from those of the other aminotransferase so far studied. Therefore, the classifications of the aminotransferases based on their primary structures, three dimensional structures, and stereochemistry of their hydrogen transfer coincide with one another. We also found that PLP-dependent amino acid racemases, the primary structures of which are similar to none of the other PLP-enzymes, catalyze the non-stereospecific hydrogen transfer on both faces of the planar intermediate. Stereospecificities for the hydrogen transfer suggest convergent evolution of the PLP-dependent enzymes. The stereochemical aspects of the enzyme reactions give a clue to the molecular evolution of the enzymes as well as the primary structures and three-dimensional structures of the enzymes.

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The β-D-glucosidase (EC. 3.2.1.21) activity of Bifidobacterium breve 203 was increased by acclimation with cellobiose, and the enzyme was purified to homogeneity from cell-free extracts of an acclimatized strain of B. breve clb, by ammonium sulfate fractionation and column chromatographies of anion-exchange, gel filtration, Gigapaite, and hydrophobic interaction. This enzyme had not only β-D-glucosidase activity but also β-D-fucosidase activity, which is specific to Bifidobacteria in intestinal flora. The molecular weight of the purified enzyme was estimated to be 47, 000-48, 000 and the enzyme was assumed to be a monomeric protein. The optimum pH and temperature of the enzyme were around 5.5 and 45°C, respectively. The enzyme was stable up to 40°C and between pH 5 and 8. The isoelectric point of the enzyme was 4.3 and the K_m values for p-nitrophenyl-β-D-glucoside and p-nitrophenyl-β-D-fucoside were 1.3 mM and 0.7 mM, respectively. This enzyme had also transferase activity for the β-D-fucosyl group but not for the β-D-glucosyl group. The N-terminal amino acid sequence of this enzyme was similar to those of β-D-glucosidase from other bacteria, actinomycetes, and plants.

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β-N-Acetylhexosaminidase (EC 3.2.1.52) was purified from the liver of a prawn, Penaeusjaponicus, by ammonium sulfate fractionation and chromatography with Sephadex G-100, hydroxylapatite, DEAE-Cellulofine, and Cellulofine GCL-2000-m. The purified enzyme showed a single band keeping the potential activity on both native PAGE and SDS-PAGE. The apparent molecular weight was 64, 000 and 110, 000 by SDS-PAGE and gel filtration, respectively. The pl was less than 3.2 by chromatofocusing. The aminoterminal amino acid sequence was NH₂-Thr-Leu-Pro-PCo-Pro-Trp-Gly-Trp-Ala-?-ASp-Gln-Gly-Val-?-Val-Lys-Gly-Glu-Pro-. The optimum pH and temperature were 5.0 to 5.5 and 50°C, respectively. The enzyme was stable from pH 4 to 11, and below 55°C. It was 39% inhibited by 10 mM HgCl₂. Steady-state kinetic analysis was done with the purified enzyme using N-acetylchitooligosaccharides (GlcNAc_n, n=2 to 6) and p-nitrophenyl N-acetylchitooligosaccharides (pNp-β-GlcNAc_n; n=1 to 3) as the substrates. The enzyme hydrolyzed all of these substrates to release monomeric GlcNAc from the nonreducing end of the substrate. The parameters of K_m and k_cat at 25°C and pH 5.5 were 0.137mM and 598s^-1 for pNp-β-GlcNAc, 0.117mM and 298s^-1 for GlcNAc₂, 0.055mM and 96.4s^-1 for GlcNAc₃, 0.044 mM and 30.1s^-1 for GlcNAc₄, 0.045 mM and 14.7 S^-1 for GlcNAc₅, and 0.047 mM and 8.3 S^-1 for GlcNAc₆, respectively. These results suggest that this β-N-acetylhexosaminidase is an exo-type hydrolytic enzyme involved in chitin degradation, and prefers the shorter substrates.

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Polar orange pigments were extracted from the cultured cells of marine bacterium strain SD-212 and purified by chromatographic methods. The structures of two new trihydroxy-keto-carotenoids, (2R, 3S, 3'S)-2-hydroxyastaxanthin (1) and (2R, 3S, 3'R)-2-hydroxyadonixanthin (2) were determined by means of spectral methods. Known carotenoids (3S, 2'R, 3'R)-erythroxanthin (3), (2R, 3S, 2'R, 3'S)-2, 3, 2', 3'-tetrahydroxy-β, β-carotene-4, 4'-dione (4), (2R, 3S, 2'R, 3'R)-2, 3, 2', 3'-tetrahydroxy-β, β-caroten-4-one (5), (3S, 3'S)-astaxanthin (6), and (3S, 3'R)-adonixanthin (7) were also isolated.

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The hypoglycemic effects of Lagerstroemia speciosa L., known by the Tagalog name of banaba in the PhilliPines, were studied using hereditary diabetic mice (TypeII, KK-A^Y/Ta Jcl). The mice were fed a test diet containing 5% of the hot-water extract (HWE) from banaba leaves, 3% of the water eluent of the partial fraction unadsorbed onto HP-20 resin of HWE (HPWE), and 2% of the methanol eluent of the partial fraction adsorbed onto HP-20 resin of it (HPME) for a feeding period of 5 weeks. The elevation of blood plasma glucose level in non-insulin dependent diabetic mice fed the cellulose as control (CEL) diet were almost entirely suppressed by addition of either HWE or HPME in place of cellulose in the CEL diet. Water intakes were inclined to increase gradually in the group fed either CEL or HPWE, but lower in the mice fed either HWE or HPME than in the animals given either CEL or HPME. The level of serum insulin and the amount of urinary excreted glucose were also lowered in mice fed HWE. Plasma total cholesterol level was also lowered in mice fed the either HWE or HPME. It is suggested that HWE, especially HPME, obtained from banaba leaves have beneficial effects on control of the level of plasma glucose in non-insulin dependent diabetes mellitus.

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OryzacystatinsI andII, cysteine proteinase inhibitors in rice seeds, caused growth retardation of different species of bean insect pests, Callosobruchus Chinensis (Coleoptera) and Riptortus clavatus (Hemiptera), when added to their diets at concentrations of 0.3-0.5% (w/w). At concentrations of up to 1%, almost all insects died. Our results suggest the usefulness of cystatin for insect pest control and also the critical role of cysteine proteinase in the digestive events of insects.

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A heteropolysaccharide and aβ-D-glucan were isolated from a lichen, Gyrophora esculenta Miyoshi (Iwatake), by cold water and alkali extractions, respectively. The chemical properties of purified polysaccharides were examined by acid hydrolysis, methylation, and GC-MS. The heteropolysaccharide is a highly branched galactomannan-type polysaccharide, containing anα-(1→6)-linked D-mannan backbone, and the glucan is a linear (1→6)-β-D-glucan. With regard to the antitumor activity, both the galactomannan and (1→6)-β-D-glucan had moderate inhibition activities on Sarcoma 180, but lower than those of branched (1→3)-β-D-glucans.

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Diacyl glyceryl ethers (DGEs) as present in marine animals were chemically synthesized and then were assessed for their oxidative and hydrolytic stabilities. CDD and CEE were easily autoxidized but not COO. The autoxidation rate of DGEs followed the unsaturation of constituent fatty acids, while alkyl chains did not affect the autoxidation of DGEs. DGEs were susceptible to hemoglobin-catalyzed oxidation as well as triacylglycerols (TGs). However, the oxidation rate of DGE was lower than that of the corresponding TG. The oxidation rate of DGEs was CEE > CDD > COO, and did not always follow the unsaturation of constituent fatty acids. DGEs with EPA and DHA were easily oxidized by lipoxygenase, and the oxidation rate of DGEs comprised of DHA Was BDD > CDD > SDD. On the other hand, DEGs were hardly hydrolyzed by porcine pancreatic lipase except for COO. These results suggest the possibility that DGE may be related to the oil quality of marine foods during storage.

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By using transposon mutants it was demonstrated that biphenyl catabolic Bph enzymes have very relaxed substrate specificities for a variety of aromatic compounds. However, the substrate ranges of the Bph enzymes of two strains used were different from each other. Pseudomonas pseudalcaligenes KF707 Bph enzymes converted biphenyls substituted with halogen, hydroxyl; methyl, and nitro groups; and biphenyl-related compounds such as biphenylmethane, dibenzyl, diphenylether, diphenylamine; and benzalacetophe-none. The same enzyme system was almost inactive for benzene derivatives. Pseudomonas Sp. KF712 Bph enzymes showed much broader substrate specificities than those of KF707, since the bphC mutant of this strain converted many benzene derivatives as well as various biphenyls and related compounds to the corresponding dihydroxy compounds.

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A tetrathionate-decomposing enzyme that catalyzes the decomposition of tetrathionate into thiosulfate and sulfate was purified to homogeneity from tetrathionate-grown Thiobacillus thiooxidans. The enzyme had an apparent molecular weight of 104, 000, and Was composed of two identical subunits (MW 58, 000)as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme had an isoelectric point at 9.6 and was most active at pH 3.0-3.5 and 40°C. Enzyme activity was increased approximately 100-fold in the presence of 400 mM sulfate ion. The Michaelis constant of this enzyme for tetrathionate in the presence of 20, 50, and 200 mM of sulfate ion was 2.4 mM. Mercuric and ferric ions completely inhibited the enzyme activity at 1 mM. Though cupric ion up to 0.01 mM markedly stimulated the activity in the presence of 20 mM sulfate ion, a higher concentration (1 mM) rather strongly inhibited the activity. Ethylenediaminetetraacetic acid (EDTA) strongly inhibited the activity, but this inhibiton was completely restored by cupric ion.

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An endo-β-N-acetylglucosaminidase was purified to homogeneity from the seeds of pea (Pisum sativum).The molecular mass of the purified enzyme was estimated to be 41, 669 Da by MALDI-TOF MS analysis and its isoelectric point to be 4.3 by isoelectric focusing. The enzyme was stable at pH 4-7 and at 25-50°C, and had the highest activity toward Man₆GlcNAc₂-PA at pH around 7.0. Oligomannose type sugar chains (Man_9-6GlcNAc₂-PA) and a hybrid type sugar chain (GlcNAc₁Man₅GlcNAc₂-PA) were most favored substrates followed by Man₅GlcNAc₂-PA, Man₃GlcNAc₂-PA, and GlcNAc₂Man₃GlcNAc₂-PA, but xylose-containing sugar chains (Man_4-3Xyl₁GlcNAc₂-PA and Man₃Fuc₁Xyl₁GlcNAc₂-PA) or a biantennary complex type sugar chain (Gal₂GlcNAc₂Man₃GlcNAc₂-PA) could not be hydrolyzed by the enzyme. The K_m values of the enzyme for Man₅GlcNAc₂-PA, Man₆GlcNAc₂-PA, and Man₉GlcNAc₂-PA were 0.40 mM, 0.25 mM, and 0.32 mM, respectively.

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By cloning and sequencing cDNA and genomic DNA and transcription initiation site mapping, the total structures including at least 1 kb of putative regulatory sequences upstream of the transcription initiation sites of three genes encoding rice sucrose synthase isoprotomers were either newly established or amended. The third type of SS gene, RSus3, has not been found in other plants. The structures of the three genes and the gene products were compared and their evolutionary sequence was proposed. Specific probes for the three SS mRNA's were developed and used for analyzing their steady state levels at different organs and under some physiological stress conditions. It appears that RSus2 is a house-keeping gene, RSus3 is highly specific to the grain, and the expression of RSus1 shows a tendency to complement that of RSus3. A possible cause of the presence of the third rice SS gene was discussed. We also reported a novel method to synthesize single-stranded DNA for S1 mapping of a transcription initiation site associated with extended secondary structures.

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Four inhibitors of glycerol-3-phosphate dehydrogenase were isolated from Ginkgo biloba and identified as anacardic acids (6-tridecylsalicylic acid, 6-[(8Z)-pentadecenyl]salicylic acid, 6-[(9Z, 12Z)-heptadecadienyl]salicylic acid, and 6-[(8Z)-heptadecenyl]salicylic acid) by instrumental analyses. Their 50% inhibitory concentrations against the enzyme were 1-3μg/ml under the standard assay conditions. Anacardic acid inhibited the enzyme non-competitively. The sarcotesta contained most of anacardic acids, and nuts a little.

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To further analyze the biodegradation of polyurethane polymers, we investigated the biodegradation of low-molecular-weight N-tolylcarbamate model compounds with structures closely resembling the urethane linkages found in polyurethanes based on tolylene-diisocyanate (TDI). Soil microflora were screened for microorganisms that were able to utilize toluene-2, 4-dicarbamic acid, diethyl ester (compound 1) as the sole source of carbon, and the soil fungus Exophiala jeanselmei strain REN-11A was selected as the most effective strain. Several N-tolylcarbamate compounds Were used, and it was found that REN-11A was able to degrade compound 1, as well as the related compound toluene-2, 6-dicarbamic acid, diethyl ester, very efficiently. Further investigation showed that compound l was biodegraded to tolylene-2, 4-diamine via the aromatic amine intermediates carbamic acid, (3-amino-4-methylphenyl)-, ethyl ester and carbamic acid, (5-amino-2-methylphenyl)-, ethyl ester.

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β-Xylosidase andα-fucosidase were purified from the viscera of apple snails (Pomacea canaliculata)using ammonium sulfate precipitation, hydrophobic chromatography on Butyl-Toyopearl, gel filtration on Sephacryl S-300, and Q-Sepharose column chromatography. β-Xylosidase andα-fucosidase are glycoproteins and their molecular masses were estimated to be approximately 85 kDa and 54 kDa by SDS-polyacrylamide gel electrophoresis, and assumed to be about 96 kDa and 230 kDa from their sedimentation coefficients, respectively, indicating that the former is a monomer and the latter has a tetrameric structure. β-Xylosidase is stable at pH 4-10 and its optimum pH toward pNP-β-D-xyloside is around 3:6, whileα-fucosidase is fairly stable at 65°C and pH 4-10, and its optimum pHs toward pNP-α-L-fucoside are around 3.2 and 5.2.β-Xylosidase released a xylose residue from Xylβ1→2Manβ1→4GlcNAcβ1→4(Fucα1→3)GlcNAc-PA and Manα1→6(Manα1→3)(Xylβ1→2)Manβ1→4GlcNAcβ1→4(Fucα1→3)GlCNAc-PA, but not from GlcNAcβ1→ 2Manα1→6(GlcNAcβ1→2Manα1→3)(Xylβ1→2)Manβ1→4GlcNAcβ1→4(Fucα1→3)GlcNAc-PA, whileα-fucosidase released a fucose residue from these three PA-oligosaccharides.

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γ-Polyglutamic acid (γ-PGA) produced by Bacillus subtilis (natto) was detected by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and basic dye staining. Using this method, the molecular weight ofγ-PGA was estimated at 275, 000. This value was almost the same in all bacterial strains tested. As other applications of this SDS-PAGE method, degradation of γ-PGA by acid and heat treatment and a cross-linking reaction with carbodiimide and ethylenediamine were made visible in acrylamide gel. In the growth curve of the bacteria, γ-PGA production was detected in early stationary phase by SDS-PAGE.

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Two lectins were purified from Helianthus tuberosus callus by maltose affinity chromatography and subsequent preparative electrophoresis. The lectins were designated HTAI and HTAII and their molecular masses were about 34 kDa by gel-filtration chromatography. A single band of 17 kDa and bands of 17 kDa and 18 kDa were detected after SDS-PAGE of HTA I and HTA II, respectively; indicating that HTAI is a homodimer while HTAII is a heterodimer. The amino acid compositions of the two lectins were very similar; they were rich in glycine residues, lacking detectable amounts of methionine, cysteine, and histidine. A hapten-inhibition assay showed that HTA I and HTA II had identical saccharide-binding specificity to the extent tested and belonged to the group of so-called mannose/glucose-binding lectins. They had high affinity forα-linked manno-oligosaccharides. Each HTA completely lost its hemagglutinatinig activity at pH 5.0, as a result of its dissociation to monomers, but it did not lose its ability to bind to oligosaccharides.

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A thermostable maltooligosyl trehalose synthase was purified from a cell-free extract of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius ATCC 33909 to an electrophoretically homogeneous state by successive column chromatography on Sepabeads FP-DA13, Butyl-Toyopearl 650M, DEAE-Toyopearl 650S, Ultrogel AcA44, and Mono Q. The enzyme had a molecular mass of 74, 000 by SQS-polyacrylamide gel electrophoresis and a pl of 5.9 by gel isoelectrofocusing. The N-terminal amino acid of the enzyme was methionine. The enzyme showed the highest activity from pH 5.0 to 5.5 and at 75°C, and was stable from pH 4.5 to 9.5 and up to 85°C. The enzyme activity was inhibited by Hg²⁺ and Cu²⁺. The Km_s of the enzyme for maltotetraose, maltopentaose, maltohexaose, maltoheptaose, and short chain amylose ((DP)^^^-18) were 41.5 mM, 7.1 mM, 5.7 mM, 1.4 mM, and 0.6 mM, respectively.

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A thermostable maltooligosyl trehalose trehalohydrolase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius ATCC 33909 was purified from a cell-free extract to an electrophoretically pure state by successive column chromatographies on Sepabeads FP-DA13, Butyl-Toyopearl 650M, DEAE-Toyopearl 650S, Toyopearl HW-55S and Ultrogel AcA44. The enzyme had a molecular mass of 59, 000 by SDS-polyacrylamide gel electrophoresis and a pl of 6.1 by gel isoelectrofocusing. The N-terminal amino acid of the enzyme was methionine. The enzyme showed the highest activity from pH 5.5 to 6.0 and at 75°C, and was stable from pH 5.5 to 9.5 and up to 85°C. The activity was inhibited by Hg⁶<2+>, Cu²⁺, Fe²⁺, Pb²⁺, and Zn²⁺. The K_m values of the enzyme for maltosyl trehalose, maltotriosyl trehalose, maltotetraosyl trehalose, and maltopentaosyl trehalose were 16.7 mM, 2.7 mM, 3.7 mM, and 4.9 mM, respectively.

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Protease production stimulating genes were isolated from a soybean protein degrading bacterium, Bacillus stearothermophilus HA19. The cloned fragment stimulated production of a 37-kDa protease in B.subtilis. The nucleotide sequence of the genes and their flanking regions were identical to the B. subtilis cell shape determinant genes mreC and mreD [J. Bacteriol., 176, 6729-6742 (1992); J. Bacteriol., 176, 6717-6728 (1992)]. The mreC and mreD genes in B. subtilis stimulate secretion of a neutral protease (37-kDa), and the protease activity in the culture medium reached 2500 U per ml (approximately 10 times higher than the host strain) after 24h of cultivation in L broth, suggesting the mreCD genes regulate protease expression and the protease is related to the cell shape determination in Bacilli. The protease productions in B. subtilis carrying mreC or mreD deletion plasmids were not elevated, so the 37-kDa protease stimulation requires both mreC and mreD genes. The extracellular protease was purified, and the molecular mass of the enzyme was 37, 000 Da by SDS-polyacrylamide gel electrophoresis and gel filtration. The optimum pH and temperature for the enzyme activity were 7.0 and 50°C, respectively, and the enzyme was stable at pH 7-10. The enzyme was inactivated by EDTA, but not by phenylmethyl sulfonyl fluoride and diisopropyl fluorophosphate.

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Synechococcus sp. PCC7942 is a phototrophic cyanobacterium. In this study we cloned a Synechococcus gene that has a striking effect on the production of the Escherichia coli outer membrane proteins, OmpC and OmpF, provided that this gene was introduced by a multicopy plasmid into the heterologous cells. This multicopy gene in E. coli cells was able to specifically shut off the production of both OmpC and OmpF at the level of transcription. The nucleotides were sequenced for this gene, named sis, and its gene product was purified from E. coli to near homogeneity. A computer-aided search found that the deduced aminio acid sequence consisting of 138 residues is novel, with no significant similarity to any other protein in the databases. Since the transcription of ompC and ompF is regulated by the regulatory factors EnvZ and OmpR, through phosphotransfer signal transduction, we explored the inhibitory effect of Sis in various genetic backgrounds as to envZ and ompR. In particular, the inhibitory effect of Sis was observed even in an ΔenvZ background, but was not observed in a certain background in which the ompC and ompF transcription was supported by a mutant OmpR that can function in a phosphorylation-independent manner. These results suggested that the EnvZ kinase may niot be the direct target of Sis, but rather that the process(es) concerning the phosphorylation and/or dephosphorylation of the OmpR protein may be affected by Sis. However, no direct effect of Sis was seen in an in vitro OmpR-phosphorylation assay with the purified OmpR and Sis proteins. Based on these results, possible functions of Sis are discussed with special reference to the phosphotransfer signal transduction in E. coli.

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Non-thermal effects of a ceramics radiation on reversibility of lactate dehydrogenase reaction have been investigated using the enzyme irradiated on cooling, and a pyruvate/NADH (system I) or a lactate/NAD⁺ (system II) as substrate. The K_m for lactate in the system II using the irradiated enzyme tended to decrease just like balancing with the increase in K_m for pyruvate in system I. The V_max/K_m for system II was increased 2.3-fold by the 18-h irradiated enzyme. Each enthalpy and entropy change in system II using the 18-h irradiation of the enzyme was decreased by 21 kJ mol^-1, although that in system I was increased by 12 kJ mol^-1. From a thermodynamic analysis, it was estimated that solvation of the active center of LDH was promoted and stabilized by the irradiation, and that it caused this regulation of the reversibility of LDH.

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A practical total synthesis of (2S, 3S, 4R)-1-O-(α-D-galactopyranosyl)-N-hexacosanoyl-2-amino-1, 3, 4-octadecanetriol (KRN7000), an antitumorial and immunostimulatory glycosphingolipid derived from agelasphins, was achieved in 14 steps starting from D-lyxose in a 16% overall yield.

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The strongest tibrinolytic protease (F-III-2) in the six enzyme proteins purified from earthworm, Lumbricus rubellus [N. Nakajima et al., Biosci. Biotech. Biochem., 57, 1726-1730 (1993)] has been modified chemically with fragmented human serum albumin (mol. wt., 10, 000-30, 000). The modified enzyme lost the antigenicity of the native enzyme and reacted with the antisera against human serum albumin, the human serum albumin fragments, and the conjugate with the natiVe enzyme to form precipitation lines, which fused with each other. The conjugate was significantly more resistant to inactivation by protease inhibitors in rat plasma. The enzyme was a non-hemorrhagic protein and did not induce platelet aggregation. The enzyme kept potent proteolytic activity for fibrin and fibrinogen than that of human plasmin. The enzyme easily solubilized actual fibrin clots (thrombi) of whole blood induced by thrombin in a rat's vena cava. The continuous fibrinolysis for fibrin suspension in an enzyme reactor system using the modified enzyme immobilized to oxirane-activated acrylic beads has been achieved without any inactivation of the activity at least for more than 1 month. The N-terminal amino acid sequence of the protein was also investigated and the sequence showed local similarity to those of the serine proteases such as plasmin and chymotrypsin.

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The following gibberellins (GAs) and antheridiogens were identified by their mass spectra and Kovats retention indices from combined gas chromatography-mass spectrometry of purified extracts of the prothallia and sporophytes of Anemia phyllitidis, a Schizaeaceous fern: a trace amount of GA₉ (4-week-old prothallid);GA₉, GA₂4, GA₂5, antheridic acid and 3-epi-GA₆3 (6-week-old prothallia); and GA₄, GA₉, GA₁5, GA₁9, GA₂0, and GA₂4 [young sporophytes (younger than one year old) and/or old sporophytes (between one- and two years old). Of these compounds, GA₂4, GA₉, and GA₄ were quantified by gas chromatography-selected ion monitorinig, using ²H-GAs as internal standards, and the content of antheridic acid, the principai antheridiogen, was evaluated by a radioimmunoassay which we have developed. The results indicate that endogenous levels of GAs and antheridiogens in prothallia began to increase rapidly between 4 and 6 weeks after sowing, the contents of anthendic acid and GA₂4, the most abundant GA in 6-week-old prothallia, being 107.4 and 37.9ng/g fresh weight, respectively. The most abundant GA in the sporophytes was GA₉, the content in young and old sporophytes being 15.3 and 7.3ng/g fresh weight, respectively.

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Endogenous gibberellinis (GAs) and GA-related compounds at two different developmental stages of apple seeds (Malus domestica cv. McIntosh) were analyzed by gas chromatography-mass spectrometry. From the seeds at ca. 10 and/or 14 weeks after full-bloom, the following GAs and 9, 15-cyclogibberellins (9, 15-cyclo-GAs) were identified by a comparison of their mass spectra and Kovats retention indices with those of authentic specimens: GA₄, GA₇, GA₉, GA₁2, GA₁7, GA₁9, GA₂0, GA₂5, GA₃4, GA₃5, GA₄5, GA₅3, GA₅4, GA₆1, GA₆2, GA₆3, GA₇0, GA₇3, GA₈0, GA₈4, GA₈8, 3-epFGA₄, 3-epFGA₅4, and 3-epFGA₆3, 9, 15-cyclo-GA₉, 1β-hydroxy-9, 15-cyclo-GA₉, 2β-hydfoxy-9;15-cyclo-GA₉, 3α-hydroxy-9, 15-cyclo-GA₉, 3β-hydroxy-9, 15-cyclo-GA9, and 11β-hydroxy-9, 15-cyclo-GA₉. The major components in the seeds at ca. 10 weeks after full-bloom were GA₄, GA₇, GA₉, GA₁7, GA₃5, GA₅4, GA₆2, GA₈0, and GA₈4, and those in the seeds at ca. 14 weeks after full-bloom were GA₁7, GA₂5, GA₄5, GA₆2, GA₆3, GA₈0, GA₈4, 9, 15-cyclo-GA₉, and 1β, 3β-, and 11β-hydroxy-9, 15-cyclo-GA₉. New GA number (GA_103-108) haVe been allocated to 9, 15-cyclo-GA₉, and 1β-, 2β-, 3β-, 3α-, and 11β-hydroxy-9, 15-cyclo-GA₉, respectively (1β- and 3α-hydroxy-9, 15-cyclo-GA₉ were identified previously as fern anteridiogens, although GA numbers have not previously been assigned to these compounds). This is the first report of the co-occurrence of GA₇3 and 9, 15-cyclo-GAs in higher plants.

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The ompC gene expression is induced by increasing temperature as well as osmotic pressure. In this study, a mutant (TD2) defective in this thermoresponse was isolated with transposon Tnl0; the mutationi was complemented by pMAN55 or pMAN56 containing micF and mapped at 48min on Escherichia coli K-12. Furthermore, a new gene (hrsA) that suppressed the mutation was cloned. Its nucleotide sequence was analyzed and it was located close to the suc operon at 16.7min corresponding to #18F11 (Kohara bank) on E. coli genome. In TD2 containing the hrsA on a multicopy plasmid, the ompC expression was induced and dependent on OmpR with increased temperature. The HrsA was found to have Enzyme IIA, IIB, and IIC domains that are homologous to Enzyme II; involved in the fructose-specific PTS (phosphotransferase system). The putative phosphorylation sites (His87 and Cys192) were also conserved in HrsA.

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Cytotoxicity and DNA single-strand breaks caused by H₂O₂were assessed by a colony formation assay and a DNA precipitation assay, respectively, with Chinese hamster V79 cells. In both assays, caffeic acid ethyl ester showed protective effects. The structure-activity relationship showed that the o-dihydroxy structure of caffeic acid ethyl ester was essential for the protective effects.

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Two microbial strains, No. 165 and No. 168, were isolated from soil as sucrose phosphorylase producers and identified as Leuconostoc mesenteroides subsp. mesenteroides and subsp. dextranicum, respectively. The sucrose phosphorylases were purified, characterized, and compared with the enzymes of L. mesenteroides AKU1102 and ATCC12291. As for the catalytic properties, these enzymes were close to each other, while as for the enzyme molecules, the No. 165 enzyme (M_r: 58, 000) was slightly diffefent from the other (M_r: 54, 000), though their N-terminal amino acid sequences were almost the same.

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The sucrose phosphorylase gene of an isolate, Leuconostoc mesenteroides No. 165, was amplified by PCR, cloned on pUC118, and expressed in E. coli. The nucleotide sequence of the gene showed 96.3% similarity to that of L. mesenteroides ATCC12291 and 67% to that of Streptococcus mutans, but low similarity to the Agrobacterium vitis gene. The cloned gene, which fusing with lacZα, was expressed inducibly with IPTG in E. coli to produce an active enzyme in large quantities that accounted for aboiit 50% of the total cell protein.

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A bioflocculant from a fungus, Aspergillus sp. JS-42, was purified by precipitations with acetone and cetylpyridinium chloride. The flocculating activity was not significantly affected by pH from 3 to 8, but was stimulated by the addition of CaCl₂, and was effective only when the reaction mixture contained an adequate amount of flocculant. The flocculant could efficiently flocculate all tested solids suspenided in aqueous solution, including various microorganisms, organic acids, and inorganic materials.

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Effects of caffeic acid and chlorogenic acid on mutagenicity were studied using the Salmonella typhimurium system. These compounds had inhibitory effects on the mutagenicity of Trp-P-1 and Glu-P-2. Caffeic acid completely eliminated the mutagenicity induced by activated Glu-P-2. Some compounds analogous to caffeic acid, such as cininamic acid, coumaric acid, and ferulic acid, also significantly decreased the mutagenicity of Glu-P-2.

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A poly(vinyl alcohol) (PVA)-degrading mixed culture that consisted of sereval microorganisms was obtained from activated sludge from a textile factory. From the culture, a component bacterial straini, BX1, that degraded PVA was isolated, and was identified as Bacillus megaterium. This strain could degrade PVA only in the cocultivation with a bacterial strain, PN19, that was isolated from the same mixed culture.

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We investigated the relative activity of N-(β-D-glucopyranosyl)-nicotinic acid to nicotinic acid as a niacin nutrient in rats and in Lactobacillus plantarum ATCC 8014. N-(β-D-Glucopyranosyl)nicotinic acid is a detoxified product or storage form of nicotinic acid that is found in plants. The relative activity of N-(β-D-gluco-pyranosyl)nicotinic acid to nicotinic acid in rats was 1/2.3, 1/2.2, 1/1.0, and 1/1.7 as indices of the body weight gain, food intake, blood NAD content, and the increased urinary excretion of niacin and its metabolites, respectively. N-(β-D-Glucopyranosyl)nicotinic acid had no niacini activity in Lactobacillus plantarum ATCC 8014.

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A gene encoding the 13-kDa prolamin polypeptide was isolated from a rice genomic library (λEMBL3) and the nucleotide sequence of an about 3-kbp EcoRI fragment was analyzed. The cloned gene (NRP33) codes for a protein composed of 156 amino acids, including a signal peptide of 19 amino acid residues and no intron is present in the genomic clone. The nucleotide sequence contains consensus TATA and CAAT boxes, and two polyadenylation signals. In addition, there are two conserved sequences named the -300 element and 10 consecutive repeats of the trinucleotide ATT in the 5'noncoding sequence.

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The distribution coefficients of glucose, maltose, and maltotriose on cation-exchange resin in Na⁺ form with a divinylbenzene content of 4% were determined in the temperature range of 5 to 60°C. The coefficients increased with decreasing temperature. The temperature dependence of the distribution coefficient was analyzed based on the swelling pressure of the resin.

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Intracellular α-D-xylosidase from Penicillium wortmannii IFO 7237 was obtained by grinding the mold with almina in phosphate buffer, and the cell-free extract was purified to a homogeneous state on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was estimated to be 290, 000 by gel filtration chromatography (Superdex 200) and 73, 000 was obtained by SDS-PAGE. The purified α-D-xylosidase had an isoelectric point at pH 5.0. The optimum activity for the enzyme was found to be at pH 6.5 and 45°C. The enzyme showed a hydrolytic activity on p-NO₂-phenyl-α-D-xylopyranoside (α-p-NPX) while methyl-α-D-xylopyranoside (α-MX) was not hydrolyzed at all. It also showed lower activity for xyloglucan oligosacchdrides. The apparent K_m values of the enzyme for α-p-NPX and isoprimeverose were 1.9 mM and 50 mM, respectively.

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Cellulase inhibitors were synthesized from cellooligosaccharides and 1-deoxynojirimycin (DNJ) by transglycosylation, using a commercial cellulase. The structures of these cellulase inhibitors were proved to be 4-O-β-cellobiosyl-DNJ, 4-O-β-D-glucopyranosyl-DNJ and 6-O-β-cellobiosyl-DNJ by the results of ¹³C- and ¹H-NMR analyses. The inhibitory activity of each inhibitor was investigated against several carboxymethylcellulases.

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A major soybean allergen, Gly m Bd 30K, is a glycoprotein. A peptide containing a sugar chain was isolated from an α-chymotrypic hydrolyzate of the allergen. The amino acid sequence analysis of the peptide showed that its sugar chain binds to the Asn¹⁷⁰ residue. Furthermore, the sugar moiety of the allergen and peptide was shown to consist of mannose, N-acetylglucosamine, fucose, and xylose at a molar ratio of 3:2:1:1. These results indicate that Gly m Bd 30K is a N-linked glycoprotein.

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An unidentified oligosaccharide was isolated from an oligomer mixture derived by alkaline borohydride treatment from glycopro-teins of Fusarium sp. M7-1. The isolated compound was identified as O-α-D-Mannopyranosyl (1→2)-D-Mannitol-6-phosphocholine by NMR and Ms spectrometry.

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Psyllid, a noxious insect, has spread everywhere in the tropical and sub-tropical regions. This insect has an habit of infesting Leucaena leucocephala. We have found that a crude enzyme of psyllid hydrolyzed mimosine, a strongly toxic substance for livestock, into 3-hydroxy-4(1H)-pyridone, pyruvic acid, and ammonia. Besides, this enzyme was also able to cleave 3-hydroxy-4(1H)-pyridone.

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A homoserine-requiring mutant, Bacillus subtilis strain No. 615, was isolated from a cytidine-producing mutant strain No. 515. The homoserine dehydrogenase activity of strain No. 615 was reduced to less than 1/50 of that of No. 515. Strain No. 615 accumulated 23.5 mg/ml cytidine in a medium containing 16% glucose, and strain No. 515 accumulated 18.8 mg/ml cytidine under the same conditions. The effects of glucose concentration on cytidine production were examined, and strain No. 615 accumulated 30.2mg/ml cytidine in a medium containing 20% glucose.

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A new lipophilic peptide was isolated from the MeOH-CH₂Cl₂ extract of adult heads of the male silkworm, Bombyx mori, by monitoring the diapause egg-inducing activity. Partial amino acid sequencing (1-55) revealed this peptide to be a unique type having a high content of lipophilic amino acids, Val, Ala, and Pro, and many repeating sequences. The compound was thus named VAP peptide.

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The brain prolylendopeptidase (PEP) inhibitory activity of syn-thetic peptides related to the bovine brain-derived PEP inhibitor, GFAP-(38-55) (MPPPLPARVDFSLAGALN), was investigated. Homologous peptides such as MPPPLPTRVDFSLAGALN (human type) and MTPPLPARVDFSLAGALN (mouse type) also inhibited PEP. Among various synthetic fragments of GFAP-(38-55), only MPPPLP had a K_i essentially the same as that of GFAP-(38-55). Similar synthetic proline-rich peptides such as synapsin fragments and SH3 domain-binding peptides had more or less inhibitory activity.

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Several phytotoxic metabolites, including a novel compound chaetoglobosini O, were isolated from Cylindrocladium floridanum Sobers et Seymore. The structure of chaetoglobosin O, including its absolute configuration, was determined by a spectroscopic analysis and chemical correlation. Purified chaetoglobosins A, C, and O showed potent growth-inhibition activity against alfalfa seedlings.

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Novel enzymatic methods were used for the measurement of the levels of total gallotannin, total amino acids, and glutamrite in green tea infusions and in canned green tea drinks. Gallotannin was measured photometrically using rhodanine and tannase. Amino acids and glutamate were measured by a fluorometric flow-analytical method using L-amino acid oxidase and L-glutamate oxidase. The analytical results showed that the canned green tea drinks examined were similar in terms of chemical composition to infusions of the Japanese green tea called sencha.

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Feeding of Tochu leaf to broiler was done to discover the role of myofibrilar protein and the effects of Tochu on meat toughness. The quality and quantity of myofibrillar protein was the same between Tochu-fed muscle and the controls. The improvement of meat quality should be concluded to be due to collagen as reported before and not due to the myofibrillar proteins.

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Exposure of 28-day-old rice seedlings for 6 days to 150 mM NaC1 was found to induce drastic decreases in relative water contents, chlorophyll, and proteins in the leaves. This effect was largely prevened when before the exposure to NaCl the seedlinigs were treated for 4 days with 15 mM glycinebetaine. Although rice plants do not accumulate glycinebetaine endogenously, added glycinebetaine was found to be taken up by the roots and to accumulate in the leaves to reach a concentration of up to 5.0 μmol per gram fresh weight. The level is comparable with those of barley and wheat, which are well known glycinebetaine accumulators, under salt stress. The quantum yield of PSII was decreased by 27% under salt stress. This decrease was also largely prevented by glycinebetaine application.

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Cell-free production of bombykol was done by incubating a pheromone gland homogenate in the presence of NADPH, ATP, and CoA. Addition of n-hexane to the reaction mixture stimulated bombykol production, resulting in production of 238 ng of bombykol from the homogenate equivalent to 2 pheromone glands after 23 h. Removal of either NADPH, ATP, or CoA resulted in no stimulation of bombykol production, suggesting that the final step of the bombykol biosynthetic pathway is done by acyl CoA synthetase and reductase, sequentially. Incubation first with ATP or high concentrations of ATP suppressed the production of bombykol. Since incubation with ATP also inhibited conversion of [1-¹⁴C]palmitoyl CoA into 1-hexadecanol, the inhibitory action of ATP seemed attributable to inactivation of the acyl CoA reductase by phosphorylation, as mediated by a protein kinase in the homogenate. Our results suggest that the activity of acyl CoA reductase in bombykol biosynthesis is regulated by phosphorylation/dephosphorylation, and that the activation occurs by dephosphorylation as mediated by phosphoprotein phosphatase.

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The flowering of Sagittaria pygmaea Miq. was promoted by 2, 6-diisopropylphenoxyacetic acid, as well as by gibberellic acid (GA₃).Uniconazole canceled the promotive effect of the phenoxyacetic acid, while prohexadione shortened the period required for flowering. Endogenous GAs seem to play an important role in the flowering of S. pygmaea, and 2, 6-diisopropylphenoxyacetic acid might affect GA biosynthesis or metabolism.

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